Natalie Eva Nieuwenhuizen

Anisakiasis is caused by ingestion of third-stage larvae (L3) of Anisakis in fish. The last decade has improved our understanding of Anisakis and its clinical manifestations, driven by advancements in high-throughput “omics” techniques and molecular diagnostics. This review summarizes major advancements, focusing on molecular mechanisms underlying its pathogenesis, host–pathogen interactions and novel diagnostic approaches. Taxonomical revisions have refined the Anisakis genus, reclassifying several species into Skrjabinisakis and Peritrachelius. Research has highlighted the critical role of Anisakis extracellular vesicles in modulating host immunity. Significant diagnostic breakthroughs include the use of the IgA/IgG4 ratio and antibody avidity profiling to differentiate gastroallergic anisakiasis from chronic urticaria. Identification of α-Gal epitopes in L3 suggests a novel link to α-Gal syndrome and red meat allergy. Ani s 13 and Ani s 14 have been identified as new major allergens, although Ani s 7 remains the gold standard for serological diagnosis. Modern systems biology is revealing how larvae adapt to their host environments, including thermal stress and glucose availability. Finally, emerging evidence suggests potential links between chronic Anisakis exposure and pathologies like cancer and sepsis. These advancements underscore the necessity of global clinical awareness and the potential for Anisakis-derived molecules as templates for future immunotherapies.

The Transforming Growth Factor beta (TGF-beta) superfamily, encompassing molecules such as TGFs, activins, Bone Morphogenetic Proteins (BMPs), Growth/Differentiation Factor (GDFs) and Nodals, represents the largest family of growth and differentiation factors, playing crucial roles in developmental and physiological processes across animal species (1). These molecules are integral to tissue homeostasis and cell fate determination. Among them, TGF-beta is particularly noted for its regulatory influence on immune responses and tissue fibrosis (2,3). Recent research has expanded our understanding of the immune functions of other superfamily members, including activin A and BMPs (4). These molecules signal through receptor complexes composed of type I and II serine/threonine kinase receptors, which activate Smads and initiate transcription. The functional diversity of TGFbeta superfamily ligands is attributed to the varied combinations of receptor complexes and the interaction of Smads with numerous transcription factors and co-regulators (5). Despite growing evidence of their roles in immune responses, the involvement of these cytokines in diseases, especially infectious diseases, remains underexplored. This gap is partly due to the embryonic lethality of gene deletions and the challenges in detecting their roles through transcriptomic approaches (6). However, advancements in genetic technologies, such as CRISPR/Cas9 and specific inhibitors, offer promising avenues to uncover novel functions of these molecules (7). This research topic aimed to elucidate the roles of TGF-beta superfamily members in immune homeostasis and disease. The primary objectives included investigating the signaling pathways of these molecules, understanding their modulation of immune responses, and exploring their involvement in various diseases. Specific questions that were addressed included the mechanisms by which these cytokines influence immune responses and their potential roles in infectious diseases, autoimmunity, allergy, inflammation, and cancer as well as the potential therapeutic targeting of TGF-beta superfamily molecules.

The standard treatment regime for drug-sensitive TB is two months of therapy with rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA), and ethambutol (EMB), followed by four months treatment with RIF and INH (2) Infection with Mtb strains resistant to RIF and INH is termed multidrug-resistant TB (MDR-TB), but RIF-mono-resistant TB is routinely treated the same as MDR-TB (3). As part of this Research topic, Birhanu et al. performed a meta-analysis on the prevalence of RIF-resistant Mtb in Ethiopia and found it was around 7%. Worldwide, around 5% of patients with TB have MDR-TB, but in some countries (Moldova, Ukraine, Kazakhstan, Kyrgyzstan) the proportion exceeds 25%. The starting treatment regimen for TB should consist of at least four drugs that are likely to be active (3,4). The WHO recommends including fluoroquinolones (FQs), bedaquiline (BDQ), and linezolid (LZD), as they are considered highly effective (34 ). TB caused by an Mtb strain which is resistant to INH, RIF and a FQ is defined as pre-extensively drug resistant TB (pre-XDR TB), while XDR TB is defined as TB caused by strains that are resistant to INH, RIF, a FQ, and either BDQ or LZD or both (4) (WHO). BDQ, delamanid (DLM) and pretomanid (PTM) were the first novel TB drugs to be developed after a gap of four decades (5,6). The remarkable efficacy of BDQ and PTM improved treatment success and led to the introduction of a 6-month short-course regimen for MDR-TB (6). Unfortunately, within years of the introduction of these novel drugs, the first clinical isolates of Mtb resistant to BDQ and DLM were identified (5). The rapid appearance of BDQ-resistant strains is disturbing. Interestingly, there is cross-resistance between BDQ and CFZ due to their shared efflux pathways, indicating that the use of CFZ could lead to BDQ resistance (7). As part of this Research Topic, Islam et al. reviewed recent studies on the mechanisms of action of BDQ and CFZ, individual resistance and cross-resistance.Antimicrobial resistance can be intrinsic, acquired, or adaptive (8,9). The formation of biofilms by Mtb is an important intrinsic factor in drug resistance, since this physical barrier protects the bacilli against antibiotics (10). Furthermore, Mtb is instrinsically resistant to many antibiotics due to its secretion of drug-modifying and degrading enzymes and its unique cell envelope, composed of long-chain mycolic acids, highly branched arabinogalactan polysaccharides and a meshwork of cross-linked, modified peptidoglycans (11). For example, Mtb is resistant to most beta-lactams due to its expression of beta-lactamase, the impermeable nature of its cell wall and its non-classical peptidoglycan cross-links (12). Some of the distinctive modifications to the peptidoglycan layer are carried out by enzymes encoded by namH and murT/gatD (12,13). Silveiro et al used CRISPR interference to silence these genes in the model organism M. smegmatis, and found that amidation of D-iso-glutamate played a role in cefotaxime and isoniazid resistance while N-glycolylation of D-iso-glutamate promoted resistance to betalactams. Thus, drugs targeting peptidoglycan modifications may be a useful avenue for developing novel TB therapies.Acquired resistance to antibiotics in bacteria occurs as a result of chromosomal genetic mutations or transfer of mobile genetic elements (11). The vast majority of drug resistant phenotypes in Mtb are due to chromosomal mutations, since there appears to be a lack of horizontal gene transfer in this species. Genetic mutations that bestow resistance include those that lead to the overexpression or alteration of the drug target, overexpression of efflux pumps or abrogation of prodrug activation. Advances in our understanding of nucleic acid biology have further revealed that adaptive epigenetic mechanisms can also contribute to drug resistance in bacteria (8). Epigenetic changes can affect gene expression without altering the DNA sequences and include changes in histone modification, DNA methylation, and expression of non-coding RNA molecules. One of the main reasons for TB relapse, drug resistance and the necessity for long therapy is the presence of bacteria known as persisters (14). After being taken up by macrophages, Mtb bacilli can enter a persistent state, in which they are dormant, which protects them from killing by antibiotics. Persisters are genetically identical to the rest of the bacterial population. It has been shown that Mtb-induced epigenetic changes can play an important role in persistence by promoting its survival in the host immune cells (15). Consequently, it has been suggested that combinatorial therapy using conventional TB drugs and agents targeting Mtb-driven epigenetic changes may improve TB treatment and reduce drug resistance (8,15).In bacteria, DNA methylation is the main epigenetic mechanism for the regulation of gene expression (16). Wu et al. set out to investigate the epigenetic features associated with resistance to EMB by inducing mono-EMB resistant Mtb in vitro, using a multi-omics approach. Fifteen genes with high methylation and low expression were identified in EMB-resistant strains, and proteomics analysis showed that the gene products of three of these (mbtB, mbtD, and celA1) were significantly downregulated. Further investigations found that expression of mbtD and celA was significantly downregulated in EMB-resistant clinical strains compared to susceptible strains. Studies suggest that celA1 inhibits biofilm formation and reduces antibiotic tolerance (17,18), while mbtD encodes a polyketide synthase required for the synthesis of mycobactins, iron chelators that scavenge iron during growth within macrophages (19). In Mycobacterium abscessus, mbtD plays a role in intracellular survival. As part of this Research Topic, Wang et al. reviewed the importance of metal ions in the survival of Mtb and suggest that targeting transcriptional regulatory proteins involved in metal ion regulation is...

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the leading cause of chronic liver disease in Western countries. Progression to metabolic dysfunction-associated steatohepatitis (MASH) occurs when fat accumulation in the liver triggers Th17 activation and other inflammatory processes. In this study, we identify the IL17A rs2275913 minor allele variant as a risk factor for fibrosis progression in MASLD patients. In patients with advanced fibrosis, we also observed an increased abundance of fungal CTG species including Candida albicans and Debaryomyces hansenii , which are potent triggers of Th17 responses. Integrating genetic risk-predisposition and mycobiome composition, we show in ex vivo T cell stimulation assays, that donors carrying the minor allele variant of IL17A rs2275913 secreted significantly higher IL-17A levels in response to CTG species. Additionally, MASH patients carrying the IL17A rs2275913 risk allele have elevated Th17/Treg ratios in peripheral blood. Taken together, our data indicate that genetic predisposition for enhanced Th17 responses in the context of mycobiome dysbiosis can trigger MASH progression and liver fibrosis. Graphical This Graphical Abstract was created with BioRender.com . Brief summary: Increased antifungal immune responses triggered by gut mycobiome dysbiosis in genetically predisposed patients can lead to severe stages of metabolic dysfunction-associated steatotic liver disease.

Candida albicans, an opportunistic fungal pathogen, produces the quorum-sensing molecule farnesol, which we have shown alters the transcriptional response and phenotype of human monocyte-derived dendritic cells (DCs), including their cytokine secretion and ability to prime T cells. This is partially dependent on the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ), which has numerous ligands, including the sphingolipid metabolite sphingosine 1-phosphate. Sphingolipids are a vital component of membranes that affect membrane protein arrangement and phagocytosis of C. albicans by DCs. Thus, we quantified sphingolipid metabolites in monocytes differentiating into DCs by High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Farnesol increased the activity of serine palmitoyltransferase, leading to increased levels of 3-keto-dihydrosphingosine, dihydrosphingosine, and dihydrosphingosine 1-phosphate and inhibited dihydroceramide desaturase by inducing oxidative stress, leading to increased levels of dihydroceramide and dihydrosphingomyelin species and reduced ceramide levels. Accumulation of dihydroceramides can inhibit mitochondrial function; accordingly, farnesol reduced mitochondrial respiration. Dihydroceramide desaturase inhibition increases lipid droplet formation, which we observed in farnesol-treated cells, coupled with an increase in intracellular triacylglycerol species. Furthermore, inhibition of dihydroceramide desaturase with either farnesol or specific inhibitors impaired the ability of DCs to prime interferon-γ-producing T cells. The effect of farnesol on sphingolipid metabolism, triacylglycerol synthesis, and mitochondrial respiration was not dependent on PPAR-γ. In summary, our data reveal novel effects of farnesol on sphingolipid metabolism, neutral lipid synthesis, and mitochondrial function in DCs that affect their instruction of T cell cytokine secretion, indicating that C. albicans can manipulate host cell metabolism via farnesol secretion. IMPORTANCE: Candida albicans is a common commensal yeast, but it is also an opportunistic pathogen which is one of the leading causes of potentially lethal hospital-acquired infections. There is growing evidence that its overgrowth in the gut can influence diseases as diverse as alcohol-associated liver disease and COVID-19. Previously, we found that its quorum-sensing molecule, farnesol, alters the phenotype of dendritic cells differentiating from monocytes, impairing their ability to drive protective T cell responses. Here, we demonstrate that farnesol alters the metabolism of sphingolipids, important structural components of the membrane that also act as signaling molecules. In monocytes differentiating to dendritic cells, farnesol inhibited dihydroceramide desaturase, resulting in the accumulation of dihydroceramides and a reduction in ceramide levels. Farnesol impaired mitochondrial respiration, known to occur with an accumulation of dihydroceramides, and induced the accumulation of triacylglycerol and oil bodies. Inhibition of dihydroceramide desaturase resulted in the impaired ability of DCs to induce interferon-γ production by T cells. Thus, farnesol production by C. albicans could manipulate the function of dendritic cells by altering the sphingolipidome.

Activin A strongly influences immune responses; yet, few studies have examined its role in infectious diseases. We measured serum activin A levels in two independent tuberculosis (TB) patient cohorts and in patients with pneumonia and sarcoidosis. Serum activin A levels were increased in TB patients compared to healthy controls, including those with positive tuberculin skin tests, and paralleled severity of disease, assessed by X-ray scores. In pneumonia patients, serum activin A levels were also raised, but in sarcoidosis patients, levels were lower. To determine whether blockade of the activin A signaling axis could play a functional role in TB, we harnessed a soluble activin type IIB receptor fused to human IgG1 Fc, ActRIIB-Fc, as a ligand trap in a murine TB model. The administration of ActRIIB-Fc to Mycobacterium tuberculosis -infected mice resulted in decreased bacterial loads and increased numbers of CD4 effector T cells and tissue-resident memory T cells in the lung. Increased frequencies of tissue-resident memory T cells corresponded with downregulated T-bet expression in lung CD4 and CD8 T cells. Altogether, the results suggest a disease-exacerbating role of ActRIIB signaling pathways. Serum activin A may be useful as a biomarker for diagnostic triage of active TB or monitoring of anti-tuberculosis therapy. IMPORTANCE: Tuberculosis remains the leading cause of death by a bacterial pathogen. The etiologic agent of tuberculosis, Mycobacterium tuberculosis , can remain dormant in the infected host for years before causing disease. Significant effort has been made to identify biomarkers that can discriminate between latently infected and actively diseased individuals. We found that serum levels of the cytokine activin A were associated with increased lung pathology and could discriminate between active tuberculosis and tuberculin skin-test-positive healthy controls. Activin A signals through the ActRIIB receptor, which can be blocked by administration of the ligand trap ActRIIB-Fc, a soluble activin type IIB receptor fused to human IgG1 Fc. In a murine model of tuberculosis, we found that ActRIIB-Fc treatment reduced mycobacterial loads. Strikingly, ActRIIB-Fc treatment significantly increased the number of tissue-resident memory T cells. These results suggest a role for ActRIIB signaling pathways in host responses to Mycobacterium tuberculosis and activin A as a biomarker of ongoing disease.

A more effective vaccine against tuberculosis than Bacille Calmette-Guérin (BCG) is urgently needed. BCG derived recombinant VPM1002 has been found to be more efficacious and safer than the parental strain in mice models. Newer candidates, such as VPM1002 Δpdx1 (PDX) and VPM1002 ΔnuoG (NUOG), were generated to further improve the safety profile or efficacy of the vaccine. Herein, we assessed the safety and immunogenicity of VPM1002 and its derivatives, PDX and NUOG, in juvenile goats. Vaccination did not affect the goats’ health in regards to clinical/hematological features. However, all three tested vaccine candidates and BCG induced granulomas at the site of injection, with some of the nodules developing ulcerations approximately one month post-vaccination. Viable vaccine strains were cultured from the injection site wounds in a few NUOG- and PDX- vaccinated animals. At necropsy (127 days post-vaccination), BCG, VPM1002, and NUOG, but not PDX, still persisted at the injection granulomas. All strains, apart from NUOG, induced granuloma formation only in the lymph nodes draining the injection site. In one animal, the administered BCG strain was recovered from the mediastinal lymph nodes. Interferon gamma (IFN-γ) release assay showed that VPM1002 and NUOG induced a strong antigen-specific response comparable to that elicited by BCG, while the response to PDX was delayed. Flow cytometry analysis of IFN-γ production by CD4⁺, CD8⁺, and γδ T cells showed that CD4⁺ T cells of VPM1002- and NUOG-vaccinated goats produced more IFN-γ compared to BCG-vaccinated and mock-treated animals. In summary, the subcutaneous application of VPM1002 and NUOG induced anti-tuberculous immunity, while exhibiting a comparable safety profile to BCG in goats.

Tuberculous granulomas are highly dynamic structures reflecting the complex host–mycobacterium interactions. The objective of this study was to compare granuloma development at the site of vaccination with BCG and its recombinant derivatives in goats. To characterize the host response, epithelioid cells, multinucleated giant cells (MNGC), T cell subsets, B cells, plasma cells, dendritic cells and mycobacterial antigen were labelled by immunohistochemistry, and lipids and acid-fast bacteria (AFB) were labelled by specific staining. Granulomas with central caseous necrosis developed at the injection site of most goats though lesion size and extent of necrosis differed between vaccine strains. CD4⁺ T and B cells were more scarce and CD8⁺ cells were more numerous in granulomas induced by recombinant derivatives compared to their parental BCG strain. Further, the numbers of MNGCs and cells with lipid bodies were markedly lower in groups administered with recombinant BCG strains. Microscopic detection of AFB and mycobacterial antigen was rather frequent in the area of central necrosis, however, the isolation of bacteria in culture was rarely successful. In summary, BCG and its recombinant derivatives induced reproducibly subcutaneous caseous granulomas in goats that can be easily monitored and surgically removed for further studies. The granulomas reflected the genetic modifications of the recombinant BCG-derivatives and are therefore suitable models to compare reactions to different mycobacteria or TB vaccines.

Tuberculosis (TB), caused by the bacillus Mycobacterium tuberculosis (Mtb), remains a leading cause of death by infectious disease, overshadowed only recently by the COVID-19 pandemic